Phase and amplitude contrast in electron microscopy of stained biological objects.

For biological objects negatively stained with heavy atom material, electron microscope images show best contrast for image detail on the scale of 10--20 A when a small objective aperture is used. In images taken under the optimum phase contrast imaging conditions of Scherzer, the required image detail is lost in unwanted noise. Both of these conditions may be described in terms of phase contrast imaging for a thin phase object. Calculations of image intensities and noise are reported for a model object consisting of heavy and light atoms randomly distributed to simulate a negatively stained protein molecule. The results are consistent with experimental observations.     

Contrast enhancement of negatively stained macromolecules and biomembranes by single sideband phase contrast interference.

A straightforward procedure is described for the production of contrast enhancement of negatively stained macromolecules and biological membranes by single sideband phase contrast interference (electron optical shadowing). The instrumental adjustment required to produce this type of phase contrast illumination is readily achieved by beam deflection from the strioscopic (dark field) mode. Part of the hollow cone of electrons from the annular condenser aperture that are unscattered by the specimen are permitted to pass through the objective aperture and interfere with the scattered beam. The electron optical shadowing effect is produced because only one side of the unscattered beam is used. Careful adjustment of the beam tilt control, with the ability to tilt in any azimuth, allows optimal illumination conditions to be achieved. The results presented show the increased image contrast obtained using as specimens the purified cylindrical macromolecule from human erythrocyte membranes, purified nuclear envelopes and collagen fibres.     

基于一种模糊逻辑算法的眼底图像增强

眼底图像是细节较多的复杂图像,在分析Pal等人提出的模糊增强算法不足的基础上,提出了一种改进的模糊增强算法,该算法简化了Pal算法中复杂的变换和逆变换运算,并采用新的增强算子,可根据不同类型的眼底图像动态调整隶属度阈值,克服了Pal算法中隶属度阈值设置为固定值的不足,而且减少了迭代次数.实验结果证明,改进后的模糊增强算法对眼底图像的增强有着更好的效果.     

应用参数化对数模型增强图像细节及对比度

传统红外图像增强方法由于未考虑人眼视觉特性而存在"超区间值",故极易丢失图像细节。本文结合对数图像处理（LIP）理论,提出了基于参数化对数模型（PLIP）的平台直方图均衡图像增强方法。该方法首先将图像变换为灰度色调函数;然后应用PLIP模型对变换后的图像进行重建,结合平台直方图均衡化来增强图像的对比度和细节信息,并利用图像评价函数（EMEE）与信息熵（En）确定模型的参数值。最后,研制了算法硬件平台,分别用中波和长波红外成像系统对算法进行了实验验证。实验结果显示：该方法对不同场景的图像均可取得很好的增强效果,图像的EMEE值比传统的平台直方图均衡算法提高至少5倍以上,对改善图像质量和视觉效果具有实用价值。     

Radiation damage in stained catalase at low temperature.

Radiation damage, which occurs in the beam of the electron microscope, has been studied in uranyl acetate stained crystals. Damage is observed in terms of the disappearance of higher orders of the electron diffraction pattern. In this study it has been found that the damage at very low specimen temperatures proceeds both more rapidly and to a greater final degree than is the case at room temperature.     

结合偏微分方程增强图像纹理及对比度

针对图像获取过程中外界光照的非均匀性会使图像存在阴影区和高亮区,从而丢失图像细节的问题,本文结合偏微分理论,提出了基于直方图均衡化的偏微分图像增强方法.该方法首先将图像变换到梯度域,通过梯度场的变换得到新的梯度函数;然后应用最小二乘原理,对梯度场中变换后的图像进行重建以增强图像的纹理细节,表现原本不清楚的细节信息.另外,针对光照的不均匀特性,结合直方图均衡化,提出反均衡变换来增强图像的亮度和对比度.提出的算法采用有限差分法将图像离散化,并与热方程相结合,其计算简单,运行速度快,具有较强的灵活性和较广的应用范围.采用Visiual C++编程,对于大小为512 pixel×512 pixel的图像的处理时间为35 ms,达到了视频实时增强显示要求.     

Enhancement of contrast in living and fixed specimens by the use of fibre optics

A method is described which enhances the contrast of living and fixed specimens examined with the stereomicroscope. It consists of immersing the ends of flexible fibre optic light sources together with the specimen in the fluid used for examination. It is reported that not only does this method increase the contrast of living specimens but that it may also be applied to specimens being prepared as thin sections or freeze fracture surfaces for examination with the transmission electron microscope. A further method of enhancement of contrast is suggested which involves the fitting of light filters of complementary colours, one to each of the fibre optic light sources, before immersion with the specimen.     

基于粗糙集与小波反锐化掩模的眼底图像增强

眼底图像是细节较多的复杂图像,为了有效去除眼底图像中的干扰噪声,提高视网膜血管的立体感和清晰度,提出了一种将粗糙集和小波反锐化掩模相结合的方法来实现眼底图像的增强.阐述了运用粗糙集中的近似及等价属性关系将图像划分成不同的区域,再根据估计好的阈值进行数据约简去除噪声的方法,又介绍了运用小波反锐化掩模法增强图像处理的方法.实验结果证明,不论在主观的视觉效果上,还是客观的噪声均方误差上,该方法对眼底图像的增强有着较好的效果.     

Three-dimensional reconstruction of single particles negatively stained or in vitreous ice.

The random-conical reconstruction method has been highly successful in three-dimensional imaging of macromolecules under low-dose conditions. This article summarizes the different steps of this technique as applied to molecules prepared with negative staining or vitreous ice, and sketches out the current directions of development. We anticipate that by using new instrumental developments, transfer function correction and computational refinement techniques, a resolution in the range of 7-10 A could ultimately be achieved.     

Enhanced contrast in electron microscopy of unstained biological material. II. Defocused contrast of large objects

A contrast minimum is observed when 88 nm diameter polystyrene latex spheres are underfocused, which is related to the wide-angle scattering peaks. Images due to scattered and non-scattered wave components are displaced due to objective-lens spherical aberration and defocus. Maximum overlap of these components produces a contrast minimum at underfocus, related to the spherical aberration of the particular lens used. Similarly, a high-contrast band at carbon-film edges arises from spherical aberration and defocus separation of non-scattered and wide-angle scattered waves. This band increases in contrast with film thickness and in width with lens defocus. These geometrical effects account for the well-known ‘blinking’ of contrast of large biological objects upon swinging through focus without an objective aperture, and for the general contrast increase of defocused large objects. Fresnel fringes account for only a narrow band of enhanced contrast at distinct edges and cannot account for contrast enhancement of large objects lacking distinct edges.     

Segmentation of epithelium in H&E stained odontogenic cysts

An algorithm for the automated segmentation of epithelial tissue in digital images of histologic tissue sections of odontogenic cysts (cysts originating from residual odontogenic epithelium) is presented. The algorithm features an image standardization process that greatly reduces variation in luminance and chrominance between images due to variations in sample preparation. Segmentation of the epithelial regions of images uses an algorithm based on binary graph cuts where graph weights depend on probabilities obtained from colour histogram models of epithelium and stroma image regions. Algorithm training used a data set of 38 images of four types of odontogenic cyst and was tested using a separate data set of 35 images of the same four cyst types. The best parameters for the segmentation algorithm were determined using a response-surface optimizer. The best parameter set resulted in an overall mean (± std. dev.) sensitivity of 91.5 ± 17% and overall mean specificity of 85.1 ± 18.6% on the training set. Particularly good results were obtained for dentigerous and odontogenic keratocysts for which the mean sensitivities/specificities were 91.9 ± 6.15%/97.4 ± 2.15% and 96.1 ± 1.98%/98.7 ± 3.16%, respectively. Our method is potentially applicable to many pathological conditions in similar tissues, such as skin and mucous membranes where there is a clear microscopic distinction between epithelium and connective tissues.     

Enhanced contrast in electron microscopy of unstained biological material. 3. In-focus phase contrast of large objects

In-focus phase contrast electron microscopy has been investigated for the enhancement of bulk contrast (i.e. the contrast of large regions) of model biological specimens. Carbon film phase plates, of measured thickness, were introduced into the back focal plane of the objective lens. Image contrast was determined from Faraday-cage intensity measurements. A contrast enhancement was observed but was measured to be less than that obtained using a very small objective aperture. This was attributed to the smaller proportion of elastic scattering and the limited spatial frequency region over which the phase contrast transfer function was uniform. Electron beam interferometry established the ability of the phase plates to preserve the coherence of the beam traversing them. Carbon granularity, of specific dimensions, was significantly enhanced by the phase plate in accordance with the phase contrast transfer function and this enhanced granularity dominated the images of biological specimens.     

Scanning electron microscopy of negatively stained catalase on a silicon wafer.

A high-resolution scanning electron microscope capable of 7 A spatial resolution at 30-kV accelerating voltage was used to observe negatively stained protein molecules. Thin platelet crystals, densely packed monolayers, and low-density deposits of beef liver catalase were prepared on the surface of silicon wafers and negatively stained with phosphotungstic acid. The tetrameric structure of the catalase molecule was observed for the first time by scanning electron microscopy on the surface of the smooth silicon wafer.